The fate of cell survival versus apoptosis is determined by the balance of anti and pro-apoptotic proteins. Expression of activator BH3-only proteins, such as BIM or tBID, leads to downstream caspase activation and apoptosis. A1 can functionally bind to and sequester BIM or tBID. An A1 inhibitor causes the release of A1-bound BIM, which activates BAX/BAK, and leads to caspase activation and more ..

Assay Overview:The fate of cell survival versus apoptosis is determined by the balance of anti and pro-apoptotic proteins. Expression of activator BH3-only proteins, such as BIM or tBID, leads to downstream caspase activation and apoptosis. A1 can functionally bind to and sequester BIM or tBID. An A1 inhibitor causes the release of A1-bound BIM, which activates BAX/BAK, and leads to caspase activation and apoptosis characterized by the release of cytochrome c from the mitochondria. Apoptosis can be confirmed as the mechanism of cell death by measuring the amount of cytochrome c released into the supernatant in this experiment. Levels of cytochrome C are determined using an Enzyme-Linked Immunosorbant Assay (ELISA) with an antibody specific for the protein.

Expected Outcome: Compounds that cause caspase activation will show an increase in absorbance in the ELISA measuring cytochrome c in the supernatant. In this cell line, which expresses the A1-2A-BIM construct similar to the primary screening line, compounds that are specific for an A1-dependent apoptotic pathway will show elevated levels of cytochrome c release.

CHL-1 cells expressing A1-2A-BIM were grown on 150 mm dishes, then washed with 1X PBS, and 3 ml of trypsin were added. After cells were dislodged, 10ml cold media were added to collect the cells. The cells were centrifuged at 1000 rpm at 4 degrees C for 4 minutes. The cell pellets were washed with cold PBS once, and resuspended in 1X AT buffer (300 mM trehalose, 10 mM HEPES-KOH pH 7.7, 10 mM KCl, 1 mM EGTA, 1 mM EDTA and 0.1% BSA), and homogenized with a Potter Elvehjem homogenizer for 30-40 strokes at 1,600 rpm on ice. After centrifuge at 600g for 10 minutes at 4 degrees C, the supernatant was centrifuged at 7000g for 10 minutes at 4 degrees C. The mitochondria pellets were carefully dislodged and re-suspended in AT buffer with 80mM KCl. The mitochondria were then aliquoted in 96 well assay plates, and incubated with appropriate compounds for 30 minutes at 30oC, then centrifuged at 8000 g for 10 minutes. Negative controls were treated with an equivalent amount of DMSO to compound treatment. Positive controls were treated with equivalent amounts of DMSO + 15 ng recombinant pro-apoptotic protein tBid. Supernatant was collected, diluted at appropriate fold into the 96 well assay kit, and detected with a human cyt c ELISA kit (R&D # SCTC0). Absorbance was read at 450nm in an M5e plate reader (Spectramax), normalized at 540 nm.

PRESENCE OF CONTROLS: Neutral control wells (NC; n=2) and positive control wells (PC; n=2) were included on the plate.EXPECTED OUTCOME: Active compounds result in increasing readout signal.NORMALIZATION:The raw signals of the plate wells were normalized using the 'Stimulators Minus Neutral Controls' method in Genedata Assay Analyzer (v7.0.3):The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.The median raw signal of the intraplate positive control wells was set to a normalized activity value of 100.Experimental wells values were scaled to this range.PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.PUBCHEM_ACTIVITY_SCORE:This was set as equal to the normalized percent activity of the 50 uM sample, rounded to the nearest integer. The compound(s) were also tested at 10 uM; the percent activity at that concentration is included here for reference.PUBCHEM_ACTIVITY_OUTCOME:Activity Outcome = 2 (active) when the PUBCHEM_ACTIVITY_SCORE was at least 10.